1. Field of the Invention
The present invention relates to Factor VIII polypeptides that are more stable than full-length Factor VIII. The present invention also relates to a method of administering the Factor VIII polypeptide to a subject to treat a blood disorder. The invention further relates to a nucleic acid construct including DNA encoding the Factor VIII polypeptide. The invention relates to a method of expressing Factor VIII in a mammal by administering the gene construct to the subject. The invention is further related to antibodies specific for the Factor VIII polypeptide.
2. General Background and State of the Art
Hemophilia A results from the quantitative or qualitative deficiency of Factor VIII (FVIII), necessitating exogenous replacement by either plasma- or recombinant-derived FVIII preparations. FVIII has a domain organization of A1-A2-B-A3-C1-C2 and is synthesized as a 2,351-amino acid single-chain glycoprotein of 280 kDa (Eaton, D. et al., 1986, Biochenlistly 25: 505–512; Toole, J. J. et al., 1984, Nature 312: 342; Vehar, G. A. et al., 1984, Nature 312: 337). Whereas the A and C domains exhibit 35–40% amino acid identity to each other and to the A and C domains of coagulation factor V, the B domain is not homologous to any known protein. Intracellular, proteolytic processing after residue Arg-1648 within the B domain generates an 80-kDa light chain (domains A3-C1-C2) and a heterogeneous-sized heavy chain of 90–200 kDa (domains A1-A2-B). The heavy and light chains are associated as a heterodimer through a divalent metal-ion-dependent linkage between the A1 and A3 domains. In plasma, FVIII circulates in an inactive form bound to von Willebrand factor (vWF) and requires proteolytic cleavage by thrombin or Factor Xa for activation (Eaton, D., et al., 1986, Biochelnistiy 25: 505–512; Girma, J. P. et al., 1987, Blood 70: 605–611; Koedam, J. A. et al., 1990, Eur. J. Biocheln. 189: 229–234). Thrombin cleavage after Arg (R) residues 372, 740, and 1689 activates FVIII coagulant activity, resulting in the complete removal of the B domain. The resulting FVIIIa heterotrimer retains the metal ion-dependent linkage between the A1 and A3-C1-C2 subunits, whereas A2 is associated with a weak affinity by electrostatic interactions (Eaton, D. et al., 1986, Biochemistiy 25: 505–512; Fay, P. J. et al., 1991, J. Biol. Chem. 266: 8957–8962; Pittman, D. D. & Kaufinan, R. J. 1988, Proc. Natl. Acad. Sci. USA 85: 2429–2433).
With an increased understanding of the biosynthesis, structure, and function of FVIII, studies have attempted to produce improved FVIII molecules for replacement therapy for patients with hemophilia A. Strategies investigated thus far have included the deletion or modification of FVIII sequences, resulting in more efficient expression. Previous studies on the requirements for functional activity of FVIII demonstrated that cleavage after Arg residues 372 and 1689 both were required for activation of FVIII and that the B domain was not required for functional activity (Eaton, D. L. et al., 1986, Biochemistty 25: 8343; Burke, R. L. et al., 1986, J. Biol. Chem 261: 12574; Toole, J. J. et al., 1986, Proc. Natl. Acad. Sci. USA 83: 5939). In order to test this hypothesis, several approaches such as the deletion, in the complementary DNA (cDNA), of large DNA fragments corresponding to domain B, giving shorter FVIII derivatives (Eaton, D. L. et al., 1986, Biocheinistry 25: 8343; Burke, R. L. et al., 1986, J Biol. Chem 261: 12574) were conducted and tested for their coagulation activity.
PCT application WO 86/06101 discloses that recombinant FVIII proteins with deletions of up to 880 amino acids in the central region still exhibit FVIII activity. In addition, Eaton et al., 1986, Biochemistoy 25:8343–8347, disclose that a polypeptide in which 766 amino acids (797 through 1562) have been deleted from the central B domain region also retains FVIII activity. These B-domain-deleted FVIII derivatives retained a site for intracellular proteolytic processing within the B domain after residue Arg-1648, which results in generation of heterogenous FVIII derivatives comprising single chain or a complex of two proteolytic cleavage products of FVIII, a 90 kDa (domains A1-A2) and an 80 kDa (domains A3-C1-C2) polypeptide. Moreover, mammalian cells transformed with a vector containing DNA encoding this deletion polypeptide had a higher production level than cells transformed with a vector containing DNA encoding the full length polypeptide. However, these B-domain deleted FVIII derivatives exhibit faster and higher activation rates by thrombin than full-length FVIII by unknown mechanisms (Eaton et al., 1986, Biochemistry 25:8343–8347; Fay et al., 1986, Biochem. Biophys. Acta 871:268–278).
U.S. Pat. No. 5,112,950 describes a FVIII derivative in which a human FVIII derivative consisting essentially of the amino acid sequence alanine-1 through aspartate-770 is linked to threonine-1667 through tyrosine-2332, wherein aspartate-770 is covalently bonded by a peptide bond to threonine-1667. A number of studies indicate that tyrosine residues at positions 346, 718, 719, 723, 1664, and 1680 are required for full activation and procoagulant activity of FVIII (Donath M. J. et al., 1995, Biochem. J 312: 49–55; Michnick D. A. et al., 1994, J Biol. Chem. 269:20095–200102). FVIII circulating in the plasma is combined with vWF, which appears to stabilize it; in effect, the half-life of FVIII in vivo decreases very rapidly in the absence of vWF (Brinkhous, K. M. et al., 1985, Proc. Natl. Acad. Sci. USA 82: 8752–8756). These studies strongly suggest that B-domain deleted Factor VIII analogs (described in U.S. Pat. No. 5,112,950, in particular), with structural alterations around 1664–1680 in the A3 region, may have potential drawbacks in terms of full activation and in vivo stability due to interference with vWF interaction. As described in U.S. Pat. No. 5,610,278, the co-expression of heavy and light chains in mammalian cells results in detectable production of FVIII. However, the combination of the two chains is inefficient, thereby decreasing the activity of the molecule (Burke, R. L. et al., 1986, J. Biol. Chem. 261, 12574; Pavirani A. et al., 1987, Biochem Biophys Res Commun. 145:234). The strategy of coexpression of heavy and light chains as a gene therapy approach in animals or humans is found to be inappropriate (Burton M et al., 1999, Proc Natl Acad Sci USA 96:12725).
U.S. Pat. Nos. 5,422,260 and 5,451,521 relate to variants of FVIII, wherein one or more of the Factor Xa, APC and thrombin cleavage sites are modified to render such sites less labile to specific proteolysis, for example, wherein one or both of the amino acids defining the cleavage site, preferably at least the arginine residues at R-740 or R-1648, is replaced by a different amino acid; and wherein the protein with deletion of amino acids from S-741 through R-1648 (fusing R-740 of the 90 kD site to E-1649 of the 80 kD site) is described but its coagulation activity was not revealed. The potential disadvantage of this modification at cleavage sites with a different amino acid is that the resultant protein would have a new epitope to potentially provoke an immunologic response. In addition, the references do not provide specified variants with internal deletion of amino acids between R-740 and R- 1689 except for the one having internal deletion of amino acids from S-741 through R-1648.
Recent studies (Chiang GG et al., 1999, Human Gene Therapy 10: 61–76) show that the B-domain deleted FVIII that is generated by deletion of amino acids from S-743 through R-1648 (fusing F-742 of N-terminal of B domain to E-1649 of the 80 kD site) which is similar to the one described in U.S. Pat. Nos. 5,422,260 and 5,451,521 exhibited only ˜50% biological activity and less specific activity and was therefore considered less suitable for therapeutic application. The reason why such a B-domain deleted FVIII possesses less biological and specific activity remains unknown. However, it is assumed that the nature of single chain FVIII with a deletion of amino acids from S-743 through R-1648 may have a different tertiary structural configuration probably due to the absence of spatial requirements between heavy chain (A1-A2) and light chain (A3-C1-C2) or to undesirable length or composition between heavy chain (A1-A2) and light chain (A3-C1-C2).
In summary, these previous strategies, although offering potential for more efficient manufacturing of recombinant protein, have not been successful. This lack of success is possibly due to their molecular characteristics such as heterogenous population of FVIII molecules, structural instability, and different thrombin activation profiles compared with that of full-length FVIII. In addition, since a variety of B-domain deleted FVIII are expressed as fused molecules, there is a possibility that the unnatural amino acid sequence (junction region of heavy chain and light chain) will remain without complete processing, and when administered into blood, possibly show a novel antigenicity (Esmon P. C., et al., 1990, Blood 76: 1593–1600, 1990). However, it is not clear whether the unnatural amino acid sequences in the fusion sites could be immunogenic, as demonstrated by the previous study (Pittman D. D. et al., 1993, Blood 81:2925). Under the circumstances, there is a desire for the development of an active and safe FVIII derivative that possesses similar profiles of thrombin activation and improved productivity.